Method of enhancing lysosomal alpha-Galactosidase A

ABSTRACT

A method of enhancing the activity of lysosomal α-Galactosidase A (α-Gal A) in mammalian cells and for treatment of Fabry disease by administration of 1-deoxy-galactonojirimycin and related compounds.

This application claims priority from U.S. patent application Ser. No.09/927,285, filed on Aug. 10, 2001, which claims priority from U.S.patent application Ser. No. 09/087,804, now U.S. Pat. No. 6,274,597,which is hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to a method of enhancing the activity oflysosomal .alpha.-Galactosidase A α-Gal A) in mammalian cells and fortreatment of glycosphingolipid storage diseases, in particular Fabrydisease, by administration of 1-deoxygalactonojirimycin and relatedcompounds.

BACKGROUND INFORMATION

Fabry disease (1) is a glycosphingolipid lysosomal storage diseasecaused by an X-linked inherited deficiency of lysosomal α-galactosidaseA α-Gal A), an enzyme responsible for the hydrolysis of terminalα-galactosyl residue from glycosphingolipids. A deficiency in the enzymeactivity results in a progressive deposition of neutralglycosphingolipids, predominantly globotriaosylceramide (ceramidetrihexoside, CTH), in vascular endothelial cells causing renal failurealong with premature myocardial infarction and strokes in patients withthis condition (2). This disorder is classified by clinicalmanifestations into two groups: a classic form with generalizedvasculopathy and an atypical variant form, with clinical manifestationslimited to heart. Recently, the atypical variant of the disease wasfound in 10% of adult male patients with unexplained left ventricularhypertrophy, increasing the estimation of frequency for the disorder(3), Like other glycosphingolipid lysosomal storage diseases, enzymereplacement therapy, gene therapy, bone marrow transplantation, andsubstrate deprivation are suggested as potential strategies for thetreatment of this disease (4). However, at the moment the only treatmentfor this disorder is symptomatic management. Therefore, development of anew therapeutic strategy for this disease is urgently needed.

Studies (5) on residual α-Gal A activity of mutant enzymes revealed thatsome of mutant enzymes have similar kinetic properties to normal α-Gal Abut with significant instability. This is considered as the case formost of atypical variant patients who generally showed higher residualα-Gal A activity than classical Fabry patients. For example (6), apurified mutant α-Gal A with a genotype of Q279E, found in a patientwith atypical variant of Fabry disease, had the same Km and Vivax as thenormal enzyme, but lost most of the enzyme activity by incubating theenzyme at pH 7.0 at 37° C. for 30 min while the normal enzyme was stableunder the same condition. Both mutant and normal enzymes were stable atpH 5.0 at 37° C. Furthermore, the majority of the mutant enzyme proteinin cells formed aggregate in endoplasmic reticulum (ER) and was quicklydegraded (7), suggesting that the deficiency of the enzyme activity inthis mutant maybe primarily caused by the unsuccessful exit of ERleading to excessive degradation of the enzyme protein. The presentinvention focuses on the aid of smooth escape of the enzyme from ER toprevent the degradation of the mutant enzyme.

SUMMARY OF THE INVENTION

The strategy of the invention is based on the following model. Themutant enzyme protein tends to fold in an incorrect conformation in ERwhere the pH is around 7. As a result, the enzyme is retarded from thenormal transport pathway from ER through the Golgi apparatus andendosome to the lysosome, but instead is subjected to degradation. Onthe other hand, the enzyme protein with a proper conformation istransported to the lysosome smoothly and remains in an active formbecause the enzyme is more stable at a pH of less than 5. Therefore, acompound which is able to induce a proper conformation in mutant enzymemay serve as an enhancer for the enzyme. The present inventors haveunexpectedly found that strong competitive inhibitors for α-Gal A at lowconcentrations enhance the mutant enzyme activity in cells, includingmutant α-Gal A gene transfected COS-I cells, fibroblasts from atransgenic mouse overexpressing mutant α-Gal A, and lymphoblasts fromFabry patients.

It is noted that while the above is believed to be the mechanism ofoperation of the present invention, the success of the invention is notdependent upon this being the correct mechanism.

Accordingly, it is one object of the present invention to provide amethod of preventing degradation of mutant α-Gal A in mammalian cells,particularly in human cells.

It is a further object of the invention to provide a method of enhancingα-Gal A activity in mammalian cells, particularly in human cells. Themethods of the present invention enhance the activity of both normal andmutant α-Gal A, particularly of mutant α-Gal A which is present incertain forms of Fabry disease.

In addition, the methods of the invention are also expected to be usefulin nonmammalian cells, such as, for example, cultured insect cells andCHO cells which are used for production of α-Gal A for enzymereplacement therapy.

Compounds expected to be effective in the methods of the invention aregalactose and glucose derivatives having a nitrogen replacing the oxygenin the ring, preferably galactose derivatives such as1-deoxygalactonojirimycin and 3,4-diepi-α-homonojirimycin. By galactosederivative is intended to mean that the hydroxyl group at the C-3position is equatorial and the hydroxyl group at the C-4 position isaxial, as represented, for example, by the following structures:

wherein R₁ represents H, methyl or ethyl; R₂ and R₃ independentlyrepresent H, OH, a simple sugar (e.g. —O-galactose), a 1-3 carbon alkyl,alkoxy or hydroxyalkyl group (e.g. CH₂OH).

Other specific competitive inhibitors for α-galactosidase, such as forexample, calystegine A₃, B₂ and B₃, and N-methyl derivatives of thesecompounds should also be useful in the methods of the invention. Thecalystegine compounds can be represented by the formula

wherein for calystegine A₃: R₁=H, R₂=OH, R₃=H, R₄=H; for calystegine B₂:R₁=H, R₂=OH, R₃=H, R₄=OH˜for calystegine B₃: R₁=H, R₂=H, R₃=OR, R₄=OH;for N-methyl-calystegine A₃: R₁=CH₃, R₂=OH, R₃=H, R₄=H; forN-methyl-calystegine B₂: R₁=CH₃, R₂=OH, R₃=H, R₄=OH; and forN-methyl-calystegine B₃: R₁=CH₃, R₂=H, R₃=OH, R₄=OH.

It is yet a further object of the invention to provide a method oftreatment for patients with Fabry disease. Administration of apharmaceutically effective amount of a compound of formula

wherein

-   -   R₁ represents H, CH₃, or CH₃CH₂;    -   R₂ and R₃ independently represent H, OH, a 1-6 carbon alkyl,    -   hydroxyalkyl or alkoxy group (preferably 1-3), or a simple        sugar;    -   R₄ and R₅ independently represent H or OH;        or a compound selected from the group consisting of        2,5-dideoxy-2,5-imino-D-mannitol, α-homonojirimycin,        3,4-diepi-α-homonojirimycin,        5-O-α-D-galactopyranosyl-α-homonojirimycin,        1-deoxygalactonojirimycin, 4-epi-fagomine, and        1-Deoxy-nojirimycin and their N-alkyl derivatives, will        alleviate the symptoms of Fabry disease by increasing the        activity of mutant α-Gal A in patients suffering from Fabry        disease. Other competitive inhibitors of α-Gal A, such as        calystegine compounds and derivatives thereof should also be        useful for treating Fabry disease.

Persons of skill in the art will understand that an effective amount ofthe compounds used in the methods of the invention can he determined byroutine experimentation, but is expected to be an amount resulting inserum levels between 0.01 and 100 μM, preferably between 0.01 and 10 μM,most preferably between 0.05 and 1 μM. The effective dose of thecompounds is expected to be between 0.5 and 1000 mg/kg body weight perday, preferably between 0.5 and 100, most preferably between 1 and 50mg/kg body weight per day. The compounds can be administered alone oroptionally along with pharmaceutically acceptable carriers andexcipients, in preformulated dosages. The administration of an effectiveamount of the compound will result in an increase in α-Gal A activity ofthe cells of a patient sufficient to improve the symptoms of thepatient. It is expected that an enzyme activity level of 30% of normalcould significantly improve the symptoms in Fabry patients, because thelow range of enzyme activity found in apparently normal persons is about30% of the average value (2).

Compounds disclosed herein and other competitive inhibitors for α-Gal Awhich will be known to those of skill in the art will be usefulaccording to the invention in methods of enhancing the intracellularactivity of α-Gal A and treating Fabry disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C. In vitro inhibition (FIG. 1A) and intracellular enhancement(FIG. 1B and FIG. 1C) of α-Gal A by alkaloid compounds. The alkaloidcompounds used were: (1) 2,5-Dideoxy-2,5-imino-D-mannitol, (2)α-Homonojirimycin, (3) 3,4-Diepi-α-homonojirimycin, (4) 5-O-α-DGalactopyranosyl-α-homonojirimycin, (5) 1-deoxygalactonojirimycin, (6)4-epi-Fagomine, (7) 1-Deoxy-nojirimycin, (Gal) Galactose. Theintracellular α-Gal A activity in COS-1 cells transfected by cDNA of amutant α-Gal A (R301Q) was assayed as described in “Methods”. (A) Theinhibition assay was performed under the Methods. IC₅₀'s of thecompounds were 1.3 mM (1), 2.6 mM (2). 2.9 μ(3), 0.62 mM (4), 4.7 nM(5), 0.25 mM (6), 0.8 mM (7), and 24 mM (Gal, galactose), respectively.

FIG. 2A-2B. Enhancement of α-Gal A by DGJ in fibroblasts derived from Tgmice (FIG. 2A) and lymphoblasts derived from Fabry patients (FIG. 2B).

FIG. 3. Time courses of enhancement of α-Gal A by DGJ in TgM fibroblasts(FIG. 3A) and lymphoblasts (FIG. 3B). The cell cultures were performedunder the Methods section. DGJ concentration added was 20 μM. Thegenotype of the human lymphoblasts was R301Q, mutant cell culturedwithout DGJ; o, mutant cell cultured with DGJ; ▴, normal lymphoblastcultured without DGJ; Δ, normal lymphoblast cultured with DGJ.

FIG. 4. DGJ concentration dependence of α-Gal A enhancement intransfected COS-1 cells (FIG. 4A), TgM fibroblasts (FIG. 4B) andlymphoblasts with a genotype of R301Q (FIG. 4C). The cells were culturedat 37° C. in Ham's F-10 medium (COS-1 cells, TgM fibroblasts) orRPMI-1640 medium supplemented with 10% FCS (lymphoblasts) containing DGJat a variable concentration for 4 days. The cDNA transfected into COS-1cells encoded a mutant α-Gal A (R301Q).

FIG. 5. DE-HNJ concentration dependence of α-Gal A enhancement intransfected COS-1 cells.

FIG. 6. Stabilization of DGJ enhanced α-Gal A in lymphoblasts. Δ, R301Qlymphoblasts cultivated without DGJ; ▴, R301Q lymphoblasts cultivatedwith DGJ.

FIG. 7. TLC analysis of metabolism of [¹⁴C]-CTH in TgN fibroblastscultured with DGJ. The TgN fibroblasts were cultured at 37° C. in Ham'sF-IO medium-10% FCS containing DGJ at 0 (lane 1), 2 (lane 2) and 20 μM(lane 3) for 4 days. After washing with the medium without DGJ,[¹⁴C]-CTH (200,000 cpm) in 2.5 ml of Opti-MEM medium (Gibco,Gaithersburg, Md. U.S.A.) was added to the cells, and incubated for 5hr. The cells were washed with 2 ml of 1% BSA and 2 ml of PBS threetimes each. The neutral glycolipids were extracted by CHC₁₃:MeOH (2:1),and purified by mild alkaline treatment, extraction with MeOH:n-hexane(1:1) and Folch extraction (19).

FIG. 8A. Determination of mRNA of α-Gal A in mutant lymphoblasts (R301Q)cultured with DGJ. The human mutant lymphoblasts (R301Q) were culturedwith or without 50 μM DGJ for 4 days. The mRNAs of α-Gal A weredetermined by a competitive RT-PCR method (15).

FIG. 8B. Western blot of mutant α-Gal A (R301Q) expressed in TgMfibroblasts. The supernatant of cell homogenate containing 10 μg proteinwas applied to SDS-PAGE, and Western blot was performed with ananti-α-Gal A antibody raised in rabbit.

FIG. 9. Percoll density-gradient centrifugation with TgM fibroblasts(FIG. 9A), TgM fibroblasts cultured with 20 μM DGJ (FIG. 9B), and TgNfibroblasts (FIG. 9C). The Percoll density-gradient centrifugation wasperformed with density markers (Sigma Chemical Co., St. Louis, Mo.,U.S.A.) as previously described by Oshima et al. (8). β-Hexosaminidase,a lysosomal marker enzyme, was assayed with4-methylumbelliferyl-β-N-actyl-D-glucosamine as substrate. Solid line,α-Gal A activity; broken line, β-hexosaminidase activity.

FIG. 10. Enhancement of α-Gal A in transfected COS-1 cells by DGJ. ThecDNA's transfected to COS-1 cells were α-Gal A's with the mutations onL166V, A156V, G373S and M296I. DGJ concentration added was 20 μM.

FIG. 11. Enhancement of α-Gal A activity by administration of DGJ to TgMmice. DGJ solutions (0.05 mM or 0.5 mM) were placed as drink sources forTgM mice (four mice as a group). After 1 week administration, the organswere homogenized for the determination of the enzyme activity. The datawere the subtraction of endogenous mouse α-Gal A activity obtained fromnon-Tg mice feeding with DGJ from the activity of TgM mice. The enzymeactivities presented were the mean values and the standard deviationswere less than 10%.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

Abbreviations used herein are set forth below for convenience: α-Gal A,human lysosomal α-galactosidase A; TgN mouse, a transgenic mouseoverexpressing normal human lysosomal α-galactosidase A; TgM mouse, atransgenic mouse overexpressing a mutant human lysosomal α-galactosidaseA with a single amino acid replacement of Arg at 301 position by Gln(R301Q); TgN fibroblast, fibroblast generated from a TgN mouse; TgMfibroblast, fibroblast generated from a TgM mouse; DGJ,1-deoxy-galactonojirimycin; DE-HNJ, 3,4-di-epi-α-homonojirimycin;pNP-α-Gal, {circumflex over (x)}-p nitrophenyl-α-D-galactoside;4-mU-α-Gal, 4-methylumbelliferyl-α-D-galactoside; FCS, fetal calf serum;PBS, phosphate-buffered saline; BSA, bovine serum albumin; TLC,thin-layer chromatography; CTH, globotriaosylceramide or ceramidetrihexoside; CDH, ceramide dihexoside; CMH, ceramide monohexoside; ER,endoplasmic reticulum.

Materials and Methods

Materials. Alkaloidal compounds were either purified from plants ofpartial chemical modified derivatives of the plant products (9). TgN andTgM mice were generated as previously reported (10, 11). TgN or TgMfibroblasts were established from TgN or TgM mouse as routine. Humanlymphoblasts were Epstein-Barr virus-transformed lymphoblast lines froma normal adult or patients with Fabry disease (6). Normal and mutantα-Gal A cDNAs for transient express in COS-1 cells were cloned asreported (12). α-Gal A for in vitro inhibition study of alkaloids wasexpressed and purified from the culture medium of Sf-9 cells infected bya recombinant baculovirus encoded normal α-Gal A gene (13). [¹⁴C]-CTHwas prepared by a combination of chemical and sphingolipid ceramindeN-deacylase reactions (14).

Methods

Cell culture. COS-1 cells, TgN and TgM fibroblasts were cultured inHam's F-10 medium supplemented with 10% FCS and antibiotics.Lymphoblasts were cultured in RPMI-1640 with 10% FCS and antibiotics.All cell cultures were carried out at 37° C. under 5% CO₂. As a modelfor fibroblasts and lymphoblasts, cells (3×10⁵ for fibroblasts and 5×10⁵for lymphoblasts) were cultured in 10 ml of the preferred medium with orwithout DGJ at 20 μM for 4 days before taken to the assay forintracellular enzyme activity.

Transient expression of α-Gal A in COS-1 cells. COS-1 cells (5×10⁵) weretransfected with 1 μg of plasmid DNA and 8 μl Lipofectamine (Gibco,Gaithersburg, Md., U.S.A.) in 1.2 ml Opti-MEM medium (Gibco) per 60-mmdish. After incubating at 37° C. for 6 hr, 1.2 ml of the same mediumcontaining 20% FCS was added and the culture was incubated overnight.After replacing the medium with 2.4 ml complete Ham's F-10 medium,alkaloid was added at an appropriate concentration, and furtherincubated for 1 day, before take to the assay for intracellular enzymeactivity.

Intracellular enzyme assay for α-Gal A. After washing withphosphate-buffered saline twice, the cells were homogenized in 200 μl ofH₂O₂ and 10 μl of the supernatant obtained by centrifugation at 10,000×gwas incubated at 37° C. with 50 μl of the substrate solution composed by6 mM 4-MU-α-Gal and 90 mM N-acetylgalactosamine in 0.1 M citrate buffer(pH 4.5) for the enzyme assay. All the data are the averages oftriplicate measurements with standard deviation less than 10%. One unitof enzyme activity was defined as one nmol of 4-methylumbelliferonereleased per hour at 37° C.

In vitro inhibition assay of α-Gal A. The enzyme activity was assayedwith pNP-α-Gal as substrate. A typical inhibition reaction was performedin a mixture of 200 nmol pNP-α-Gal, appropriate enzyme and inhibitor ina total volume of 120 μl with 0.05 M citrate buffer (pH 4.5). Afterincubation at 37° C. for 15 min, the reaction was terminated by additionof 1 ml of 0.2 M borate buffer (pH 9.8), and the amount of pNP releasedwas measured as the absorbance at 490 nm.

EXAMPLE 1

A series of plant alkaloids (scheme 1, ref. 9) were used for both invitro inhibition and intracellular enhancement studies of α-Gal Aactivity. The results of inhibition experiments are shown in FIG. 1A.

Among the tested compounds, 1-deoxy-galactonojirimycin (DGJ, 5) known asa powerful competitive inhibitor for α-Gal A, showed the highestinhibitory activity with IC₅₀ at 4.7 nM, α-3,4-Di-epi-homonojirimycin(3) was an effective inhibitor with IC₅₀ at 2.9 μM. Other compoundsshowed moderate inhibitory activity with IC₅₀ ranging from 0.25 mM (6)to 2.6 mM (2). Surprisingly, these compounds also effectively enhancedα-Gal A activity in COS-1 cells transfected with a mutant α-Gal A gene(R301Q), identified from an atypical variant form of Fabry disease witha residual α-Gal A activity at 4% of normal. By culturing thetransfected COS-1 cells with these compounds at concentrations cat3-10-fold of IC₅₀ of the inhibitors, α-Gal A activity was enhanced1.5-4-fold (FIG. 1C). The effectiveness of intracellular enhancementparalleled with in vitro inhibitory activity while the compounds wereadded to the culture medium at 10 μM concentration (FIG. 1B).

EXAMPLE 2

DGJ, the strongest inhibitor in vitro and most effective intracellularenhanced, was chosen for more detailed characterization. DGJ was addedto the TgM or TgN fibroblasts (FIG. 2A) and lymphoblasts derived fromFabry patients with genotypes of R301Q or Q279E mutations (FIG. 2B). Theenzyme activity found in TgM fibroblasts increased 6-fold byco-cultivation with 20 μM DGJ and reached 52% of normal. The DGJ alsoshowed a similar effect on lymphoblasts in which the residual enzymeactivity was enhanced by 8- and 7-fold in R301Q and Q279E, i.e., 48% and45% of normal. The enzyme activity in Tg normal (TgN) fibroblasts andnormal lymphoblasts also showed an increase by cultivation with DGJ.

EXAMPLE 3

The TgM fibroblasts and human lymphoblasts of normal and patient with amutation on R301Q were cultured in the presence of DGJ at 20 μM. In theculture without DGJ, the α-Gal A activity in TgM fibroblasts or mutantlymphoblasts was unchanged (FIG. 3). However, by including DGJ, theenzyme activity showed significantly increase in these cell cultures.The enzyme activity in mutant lymphoblasts reached to 64% of those foundin normal lymphoblasts cultured without DGJ at the fifth day. The enzymeactivity in normal lymphoblasts was also enhanced 30% after cultivationwith DGJ.

EXAMPLE 4

DGJ concentration dependence of α-Gal A enhancement in transfected COS-1cells, TgM fibroblasts and lymphoblasts with a phenotype of R301Q wasexamined.

As shown in FIG. 4, the enzyme activity increased with the increase inthe concentration of DGJ in the range of 0.2-20 μM in transfected COS-1cells (FIG. 4A) and lymphoblasts (FIG. 4Q, and between 0.2-200 μM n TgMfibroblasts (FIG. 4B), respectively. A higher concentration of DGJsuppressed the enhancement effect.

DE-HNJ showed the same effect on the enhancement of α-Gal A in COS-1cells transfected with a mutant cDNA of the enzyme (R301Q) at the higherconcentrations (1-1000 μM) compared with DGJ (FIG. 5). It is clear thatDE-HNJ at 1 μM in culture medium did not inhibit intracellular enzymeactivity of COS-1 cells.

EXAMPLE 5

FIG. 6 shows an experiment to measure stabilization of DGJ enhancedα-Gal A in lymphoblasts. The cells were cultured at 37° C. in 10 mlRPMI-1640 medium supplemented with 10% FCS containing DGJ at 20 μM for 4days, and 5×10⁵ cells were transferred to 13 ml of RPMI1640 with 10% FCSwithout DGJ. Two ml of the medium was taken, each day for the enzymeassay. The initial surplus of the total α-Gal A activity betweenpre-cultivation with and without DGJ was maintained for 5 days afterreplacement of the medium without DGJ (FIG. 6), suggesting that theenhanced enzyme is stable in the cells for at least 5 days.

EXAMPLE 6

To study the functioning of the enhanced enzyme in the cells, [¹⁴C]-CTHwas loaded to the culture of TgN fibroblasts.

The determination of glycolipid was performed by thin-layerchromatography using CHC1₃:MeOH:H₂O (65:25:4) as a developing solvent,and visualized by a Fuji-BAS imaging system (FIG. 7). The amount ofceramide di-hexoside (CDH), a metabolic product of CTH by α-Gal A, wascomparable between the cells cultivated with 20 μM DGJ and without DGJ(4.5% vs. 4.3% of the total neutral glycolipids), indicating that theintracellular enzyme is not inhibited by DGJ at the concentration used.

EXAMPLE 7

In order to determine whether DGJ affects the biosynthesis of α-Gal A,the level of α-Gal A mRNA in mutant lymphoblasts (R301Q) cultured withDGJ were measured by a competitive polymerase chain reaction (PCR)method (15). FIG. 8A clearly shows that the mRNA of α-Gal A wasunchanged by cultivation of lymphoblasts with 50 μM of DGJ.

On the other hand, Western blot analysis indicated a significantincrease of the enzyme protein in TgM fibroblasts, and the increasecorresponded to the concentration of DGJ (FIG. 8B). More enzyme proteinwith lower molecular weight (ca. 46 kD) in the cells cultivated with DGJsuggested the higher level of matured enzyme (16). These resultsindicate that the effect of DGJ on enhancement of the enzyme is apost-transcriptional event.

EXAMPLE 8

To confirm the enhanced enzyme is transported to the Iysosome, asub-cellular fractionation was performed with Tg mice fibroblasts (FIG.8). The overall enzyme activity in TgM fibroblasts was lower and elutedwith a density marker of 1.042 g/ml which contained Golgi apparantsfractions (20) (FIG. 9A). By cultivation with 20 μM DGJ, the enzymeactivity in TgM fibroblasts showed higher overall enzyme activity andthe majority of the enzyme eluted with the same fraction of a lysosomalmarker enzyme, β-hexosaminidase (FIG. 9B). The elution pattern of α-GalA activity in TgM was also changed to those found in TgN fibroblasts(FIG. 9C).

EXAMPLE 9

The genotypes of R301Q and Q279E were found from patients with atypicaltype of Fabry disease. The effectiveness of DGJ on enhancement of α-GalA activity was examined with other genotypes and phenotypes of Fabrydisease. In this experiment, three mutant α-Gal A cDNA's, LI66V, A156Vand G373S found in patients with classical type of Fabry disease and amutation of M296I found from patients with atypical form of the diseasewere used. FIG. 10 shows that the inclusion of DGJ increased enzymeactivity in all four genotypes tested, especially for LI 66V (7-foldincrease) and A156V (5-fold increase). The data indicated that thisapproach is useful not only for the atypical form, but also classicalform of the disease.

EXAMPLE 10.

DGJ was administrated to Tg mice by feeding 0.05 or 0.5 mM DGJ solutionsas drinking source for a week corresponding to the dosage of DGJ atapproximate 3 or 30 mg per kilogram of body weight per day. The enzymeactivity was elevated 4.8- and 18-fold in heart, 2.0- and 3.4-fold inkidney, 3.1- and 9.5-fold in spleen and 1.7- and 2.4-fold in liver,respectively (FIG. 11). The increase of the enzyme activity in organsresponded to the increase of DGJ dosage. Since the mutant gene (R301Q)was found in atypical variant type Fabry patients which have clinicalsymptoms limited to heart, the fact that oral adiministration of DGJspecifically enhances the α-Gal A activity in the heart of TgM mouse isparticularly significant.

Discussion

It is known that the ER possesses an efficient quality control system toensure that transport to the Golgi complex is limited to properly foldedand assembled proteins, and the main process of the quality control isenforced by a variety of chaperons (17). One explanation of the resultspresented in the present application is as follows: In some phenotypesof Fabry disease, the mutation causes imperfect, but flexible folding ofthe enzyme, while the catalytic center remains intact.: Inhibitorsusually have high affinity to the enzyme catalytic center, and thepresence of the inhibitor affixes the enzyme catalytic center andreduces the flexibility of folding, perhaps leading to the “proper”conformation of the enzyme. As a result, the enzyme could be passedthrough the “quality control system”, and transported to Golgi complexto reach maturation. Once the enzyme is transported to lysosome wherethe pH is acidic, the enzyme tends to be stable with the sameconformation, because the enzyme is stable under the acidic condition(6). In such cases, the inhibitor acts as chaperon to force the enzymeto assume the proper conformation. We propose to use “chemical chaperon”as a term for such low molecular weight chemical with such functions.

It is crucial for the functioning of the enzyme that the smoothdissociation of the compound from the enzyme catalytic center inlysosome could be taken. Since the compounds used in this study arecompetitive inhibitors, the dissociation of the inhibitors depends upontwo factors: i) the inhibitor concentration, and ii) pH. Dale et al.(18) have shown that binding of 1-deoxynojirimycin to α-glucosidase ispH dependent where the inhibitor bound to the enzyme 80-fold moretightly at pH 6.5 compared to pH 4.5, suggesting that the nojirimycinderivatives function as an unprotonated form. This may explain theresults on the functioning of α-Gal A in cells shown in FIG. 7, becausethe inhibitor can bind to the enzyme at neutral condition, and releasefrom the enzyme at the acidic condition where DGJ tends to beprotonated.

The results described herein show that DGJ can effectively enhancemutant α-Gal A activities in lymphoblasts of patients with atypicalvariant of Fabry disease with genotypes of R301Q and Q279E. Theeffectiveness of DGJ on other phenotypes of Fabry mutation includingclassical and atypical forms has also been examined. DGJ effectivelyenhanced the enzyme activity in all three genotypes of cell strainsderived from patients diagnosed as atypical Fabry disease, and some ofthe cell strains with classical Fabry forms having high residual enzymeactivity. According to the present invention, a strategy ofadministrating an α-Gal A inhibitor should prove to-be an effectivetreatment for Fabry patients whose mutation occurs at the site otherthan catalytic center, and also should be useful for otherglycosphingolipid storage diseases.

References cited herein are hereby incorporated by reference and arelisted below for convenience:

1. R. O. Brady, A. E. Gal, R. M. Bradley, E. Martensson, A. L. Warshaw,and L. Laster, N. Engl. J. Med. 276, 1163 (1967).

2. R. J. Desnick, Y. A. Ioannou, and C. M. Eng, in The Metabolic andMolecular Bases of Inherited Disease, eds. C. R. Scriver, A. L. Beaudet,W. S. Sly, and D. Valle (McGraw-Hill, New York), pp. 2741 (1995).

3. S. Nakao, T. Takenaka, M. Maeda, C. Kodama, A. Tanaka, M. Tahara, A.Yoshida, M. Kuriyama, H. Hayashibe, H. Sakuraba, and H. Tanaka, N. Engl.J. Med. 333, 288 (1995).

4. E. Beutler, Science 256, 794 (1992); F. M. Piatt, G. R. Neises, G.Reikensmeier, M. J. Townsend, V. H. Perry, R. L. Proia, B. Winchester,R. A. Dwek, and T. D. Butters, Science 276, 428 (1997).

5. G. Romeo, M. D'Urso, A. Pisacane, E, Blum, A. de Falco, and A.Ruffilli, Biochem. Genet. 13, 615 (1975); D. F. Bishop, G. A. Grabowski,and R. J. Desnick, Am. J. Hum. Genet 33, 71A (1981).

6. S. Ishii, R. Kase, H. Sakuraba, and Y. Suzuki, Biochem. Biophys. Res.Comm. 197, 1585 (1993).

7. S. Ishii, R. Kase, T. Okumiya, H. Sakuraba, and Y. Suzuki, Biochem.Biophys. Res. Comm. 220, 812 (1996).

8. A. Oshima, K. Yoshida, K. Itoh, R. Kase, H. Sakuraba. and Y. Suzuki,Hum Genet 93. 109 1994).

9. N. Asano, K. Oseki, H. Kizu, and K. Matsui, J. Med. Chem. 37, 3701(1994); N. Asano, M. Nishiba, H. Kizu, K. Matsui, A. A. Watson, and R.J. Nash, J. Nat. Prod. 60, 98 (1997).

10. M. Shimmoto, R. Kase, K. Itoh, K. Utsumi, S. Ishii, C. Taya, H.Yonekawa, and H. Sakuraba, FEBS Lett 417, 89 (1997).

11. S. Ishii, R. Kase, H. Sakuraba, C. Taya, H. Yonekawa, T. Okumiya, Y.Matsuda, K. Mannen, M. Tekeshita, and Y. Suzuki, Glycoconjugates J. inpress (1998).

12. T. Okumiya, S. Ishii, T. Takenaka, R. Kase, S. Kamei, H. Sakuraba,and Y. Suzuki, Biochem. Biophys. Res. Comm. 214, 1219 (1995)

13. S. Ishii, R. Kase, H. Sakuraba, S. Fujita, M. Sugimoto, K. Tomita,T. Semba, and Y. Suzuki, Biochim. Biophys. Acta 1204, 265 (1994).

14. S. Neuenhofer, G. Schwarzmann, H. Egge, and K. Sandhoff,Biochemistry 24, 525 (1985); S. Mitsutake, K. Kita, N. Okino, and M.Ito, Anal. Biochem. 247, 52 (1997).

15. G. Gilliland, S. Perrin, K. Blanchard, and H. F. Bunn, Proc. Natl.Acad. Set USA 87, 2725 (1990); TaKaRa Bio Catalog Vol. 1, D-59 (1997).

16. P. Lemansky, D. F. Bishop, R. J. Desnick, A. Hasilik, K. Von Figura,J. Biol. Chem. 262, 2062 (1987).

17. S. M. Hurtley, and A.Helenius, Annual Rev. Cell Biol. 5, 277 (1989).

18. M. P. Dale, H. E. Ensley, K. Kern, K. A. R. Sastry and L. D. Byers,Biochemistry 24, 3530 (1985).

19. Folch et al. J. Biol. Chem. 226:497 (1957).

20. Fleisher, S. and M. Kervina, Methods in Enzymology 31, 6 (1974).

It will be appreciated that various modifications may be made in theinvention as described above without departing from the scope and intentof the invention as defined in the following claims wherein:

1. A method of enhancing the activity of lysosomal α-galactosidase A in mammalian cells comprising administering an effective amount of a compound selected from the group consisting of 2,5-dideoxy-2,5-imino-D-mannitol, 3,4-diepz-α-homonojirimycin, 5-O-α-D-galactopyranosyl-α-homonojirimycin, 1-deoxygalactonojirimycin, 4-epi-fagomine, calystegine A3, calystegine B2, and calystegine B3, and N-alkyl derivatives thereof.
 2. The method of claim 1 wherein the lysosomal α-galactosidase A is a mutant form which is present in patients with Fabry disease.
 3. The method of claim 1 wherein said cells are human cells.
 4. The method of claim 3 wherein said cells are the cells of a patient with Fabry disease.
 5. A method of treating Fabry disease comprising administering an effective amount of a compound selected from the group consisting of 2,5-dideoxy-2,5-imino-D-mannitol, 3,4-diepi-α-homonojirimycin, 5-O-α-D-galactopyranosyl-α-homonojirimycin, 1-deoxygalactonojirimycin, 4-epi-fagomine, calystegine A3, calystegine B2, and calystegine B3, and N-alkyl derivatives thereof.
 6. The method of claim 5 wherein said compound is 1-deoxygalactonojirimycin or 3,4-diepi-α-homonojirimycin.
 7. The method of claim 6 wherein said compound is 1-deoxygalactonojirimycin.
 8. (canceled)
 9. A method of treating Fabry disease comprising administering an effective amount of a compound of the formula

wherein R₁ represents H, —CH₂—or CH₂OH; R₂ represents H, OH or —O-galactose; R₃ and R₄ independently represent H, or OH; R₅ represents H, or —CH₂—; R₆ represents CH₂OH, or OH; and R₇ represents H or an alkyl group containing 1-3 carbon atoms, provided that when either R₁, or R₅ is —CH₂—, they are identical and are linked to form a second ring structure. 